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The increasing sophistication of recombinant DNA techniques has provided significant progress in understanding the mechanisms involved in regulating eukaryotic gene expression. This is greatly facilitating research and development in the plant, agricultural, medical and veterinary industries. Transcription factors are an important component in the control of gene expression. However, despite their importance, mammalian transcription factors have not been well investigated for their diagnostic and therapeutic potential.
RNA polymerases in eukaryotic cells cannot initiate transcription alone; before transcription can begin, they require interaction between transcription factors and the promoter. These factors assemble at the promoter and, via a series of steps, facilitate both the binding of RNA polymerase II to the promoter and its subsequent phosphorylation and release to initiate transcription.
In addition to these general transcription factors, many thousands of transcription activators and/or negative regulators (inhibitors) exist, which control the process of initiation of gene transcription from great distances along the DNA. These factors influence the timing and extent of transcription of a particular gene. Indeed, they control whether and to what extent a particular gene is transcribed in a cell of a particular tissue type. Although most gene regulators identified to date have been found to be proteins, some transcription factors may also be RNA molecules.
In Drosophila, the transcription factor known as “Grainyhead” regulates key developmental process in the embryo and is encoded by the gene grainyhead. During development, Grainyhead is initially involved in dorsal/ventral and terminal patterning of the newly fertilized embryo through the formation of multi-protein complexes that repress transcription from the decapentaplegic, tailless and zerknuellt genes (Huang et al., Genes Dev. 9: 3177-3189, 1995; Liaw et al., Genes Dev. 9: 3163-3176, 1995). Later, grainyhead is predominantly expressed in the embryonic central nervous system in cuticle-producing tissues, where it binds to promoters and influences transcription from other developmentally regulated genes including engrailed, fushi tarazu and Ultrabithroax (Bray et al., Genes Dev. 3: 1130-1145, 1989; Dynlacht et al., Genes Dev. 3: 1677-1688, 1989; Biggin and Tjian, Cell 53: 699-711, 1988; Soeller et al., Genes Dev. 2: 68-81, 1988; Dynlacht et al., Cell 56: 563-576, 1991; Attardi and Tjian, Genes Dev. 7: 1341-1353, 1993; Uv et al., Mol. Cell Biol. 14: 4020-4031, 1994).
The importance of grainyhead in Drosophila development is emphasised by the embryonic lethal phenotype observed in flies carrying mutations in this gene. The embryos have flimsy cuticles, grainy and discontinuous head skeletons and patchy tracheal tubes (Bray and Kafatos, Genes Dev. 5: 1672-1683, 1991). A neuroblast-specific isoform of the protein, arising from alternate splicing, has also been identified. A mutation that abolishes this isoform is pupal- and adult-lethal, and flies demonstrate uncoordinated movements (Uv et al., Mol. Cell Biol. 17: 6727-6735, 1997).
Mammalian homologs of grainyhead have previously been proposed, including three genes designated CP2, LBP-1a and LBP-9. Studies have implicated them in a wide variety of cellular and developmental events including T cell proliferation, globin gene expression and steroid biosynthesis (Sueyoshi et al., Mol. Cell Biol. 15: 4158-4166, 1995; Jane et al., EMBO J. 14-97-105, 1995; Volker et al., Genes Development 11: 1435-1446, 1997; Zhou et al., Mol. Cell Biol. 20: 7662-7672, 2000). However, in situ analyses of both CP2 and LBP-1a reveal ubiquitous expression of both genes, unlike the highly restricted pattern observed with grainyhead in Drosophila (Bray et al., 1989, supra; Dynlacht et al., 1989, supra; Bray and Kafatos, 1991, supra; Ramamurthy et al., J. Biol. Chem. 276: 7836-7842, 2001). It is concluded, therefore, that these genes are not close homologs of grainyhead.
Abnormalities in mammalian transcription factor expression are considered to play a role in a number of different genetic disorders and birth defects such as spina bifida and anencephaly. There is therefore a need to identify mammalian transcription factors and in particular close mammalian homologs of Grainyhead and to use these to develop a range of diagnostic and therapeutic agents.